组成图示
示意图生成中
传感器类型
表面等离子共振(SPR)生物传感器
检测对象
噬菌体抗体 B8、H6、C1（phage antibodies B8/H6/C1，M13噬菌体展示scFv），样品基质：PBS缓冲液（pH 7.4）
检测原理
该传感器基于表面等离子共振（SPR）光学换能。金膜表面经二硫二乙醇酸（DDA）自组装形成单分子层，半抗原B、H、C固定于DDA层，BSA封闭非特异性位点。当含噬菌体抗体B8、H6、C1的PBS样品流过传感面时，噬菌体表面展示的抗体片段与对应半抗原发生特异性结合。结合事件使金膜界面质量与局部折射率增加，导致表面等离子体共振条件改变，反射光强度-波长曲线出现共振波长位移（Δλ）。Δλ大小与结合量及亲和力相关，可实时监测结合与解离过程，从而获得动力学参数。检测无需标记，结合后以低pH柠檬酸再生传感面。
检测灵敏度
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效应效果
SPR传感器可特异性识别对应噬菌体抗体，BSA封闭后无波长漂移。10 μmol/L噬菌体抗体重复11次，RSD为1.5%。与ELISA相比，SPR在2 h内完成，ELISA需>24 h；10 μmol/L下B8、H6、C1的Δλ为1.4、1.2、0.8 nm，OD450为1.5、1.3、0.6。传感面可用0.3 mol/L柠檬酸（pH 2.7）再生。Se-B8、Se-H6、Se-C1的GPX活性为3012、2102、694 U/μmol，KD为14、12、12 μmol/L。作者认为该方法简便、快速、低成本，无需HRP标记，适用于噬菌体抗体筛选和分子相互作用动力学研究。
传感器的构成
- 基底/换能器：直角玻璃棱镜上的可更换玻璃芯片，真空蒸镀50 nm金膜（Au），产生表面等离子共振。
- 自组装修饰层：二硫二乙醇酸（DDA）在金表面形成自组装单分子层，提供结合位点。
- 捕获识别层：半抗原GSH-S-DNP-Bu（B）、GSH-S-DNP-He（H）、GSH-S-DNP-cHe（C）固定于DDA层，特异性捕获对应噬菌体抗体。
- 封闭层：5 g/L牛血清白蛋白（BSA）封闭非特异性结合位点。
- 信号标记物：无外源标记，噬菌体抗体结合质量直接产生SPR信号。
- 信号读出：SPR光学系统监测反射光强度-波长曲线及共振波长位移（Δλ）。
中文摘要
谷胱甘肽过氧化物酶（GPX）是清除活性氧的重要抗氧化酶。为获得人源化GPX催化抗体，本研究以噬菌体展示人抗体库为材料，经多轮筛选获得新型抗体。以三种S-2,4-二硝基苯基谷胱甘肽酯半抗原GSH-S-DNP-Bu（B）、GSH-S-DNP-He（H）和GSH-S-DNP-cHe（C）为靶标，经ELISA四轮筛选，得到具有谷胱甘肽（GSH）结合位点的噬菌体抗体B8、H6和C1，并用表面等离子共振（SPR）生物传感器表征。金膜经二硫二乙醇酸（DDA）修饰后，半抗原自组装固定形成传感膜，可特异性结合相应抗体；SPR实时监测共振波长变化，研究结合动力学。为提高选择性，化学修饰将硒半胱氨酸（Sec）引入B8、H6和C1，得到Se-B8、Se-H6和Se-C1，GPX活性分别为3000、2000和700 U/μmol。与ELISA相比，SPR方法更快速、简便。
英文摘要
Glutathione peroxidase (GPX) is an important antioxidant enzyme, which plays an important role in scavenging reactive oxygen species. To obtain humanized GPX catalytic antibodies, the phage displayed human antibody library on the surface of the filamentous bacteriophage was used to select novel antibodies by repetitive screening. Phage antibodies B8, H6 and C1 with the GSH-binding site were obtained from the library by enzyme-linked immunosorbent assay (ELISA) analysis with four rounds of selection against three haptens, S-2,4-dinitrophenyl t-butyl ester [GSH-S-DNP-Bu (B)], S-2,4-dinitrophenyl t-hexyl ester [GSH-S-DNP-He (H)] and S-2,4-dinitrophenyl cycle-hexyl ester [GSH-S-DNP-cHe (C)], and characterized using surface plasmon resonance (SPR) biosensor. The gold layer was modified by dithiodiglycolic acid (DDA) and three haptens were easily attached to DDA by self-assembling to form a biosensor membrane. The membrane bounds specifically corresponding antibodies. The kinetic process of the reaction between phage antibodies and their haptens was studied by SPR biosensor. In order to improve selectivity, chemical modification was used to incorporate directly catalytic group selenocysteine (Sec) into selected phage clone B8, H6 and C1 to form Se-B8, Se-H6 and Se-C1, respectively. The GPX activities of Se-B8, Se-H6 and Se-C1 were found to be 3000, 2000 and 700units/mumol, respectively. Compared with conventional ELISA analysis, the proposed method based on SPR biosensor is much more rapid and simpler.